Production of extracellular protease by a Brazilian strain of Beauveria bassiana reactivated on coffee berry borer, Hypothenemus hampei

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Production of extracellular protease by a Brazilian strain of Beauveria bassiana reactivated on coffee berry borer, Hypothenemus hampei

Eliana Tiemi Ito; Geni Varéa-PereiraI, ^*; Dalva Tomoe Miyagui^I; Maria Helena Pimenta Pinotti^I; Pedro Manoel Oliveira Janeiro Neves^II

^IDepartamento de Bioquímica e Biotecnologia

^IIDepartamento de Agronomia; Universidade Estadual de Londrina; Campus Universitário; C. P. 6001; 86051-990; Londrina - PR - Brasil

ABSTRACT

Studies were carried out on extracellular protease production by Beauveria bassiana CG432 in liquid medium containing glucose and yeast extract. B. Bassiana presented active growth after lag period of 24 h., produced 80% of the total of the extracellular protease activity in 48 h which was maximum on the 5^th culture day. The extracellular protease presented optimum activity at 60ºC, was stable up to 1M Cl^-, maintained the stability during 15 day at 4ºC and -18ºC, but was not stable if frozen repeatedly.

Key words: Protease activity, Beauveria bassiana, enzyme stability

RESUMO

A cepa brasileira do fungo entomopatogênico Beauveria bassiana CG432 foi cultivada em meio de cultivo líquido contendo glucose e extrato de levedura a 28°C, 150 rpm, durante 9 dias utilizando 1% de inóculo constituído por 10⁸conídios/mL pré-ativados em insetos vivos de broca-do-café. Avaliou-se diariamente o crescimento do fungo através da quantificação da biomassa e a atividade de proteases extracelulares no filtrado do cultivo pela determinação de peptídeos solúveis liberados pela hidrólise de soro albumina bovina a 37°C durante 30minutos. O fungo cresceu ativamente após reduzido período lag de 24 horas, produziu 80% da atividade total de proteases em 48 horas e atingiu produção máxima em 5 dias de cultivo, sem adição de substratos indutores no meio de cultivo. As proteases apresentaram atividade ótima a 60ºC, estabilidade até 1M de íons cloreto, mantiveram mais de 80% de atividade durante 15 dias a 4ºC e –18ºC, porém foram sensíveis a congelamentos repetitivos.

INTRODUCTION

The entomopathogenic fungus Beauveria bassiana is commonly isolated from dead insects in the environment (Boucias and Pendland, 1998; Cottrell and Shapiro-Ilan, 2003) and its spores are frequently used as biological control agent against pest infestation in agriculture (MacLeod, 1954; Furlong and Pell, 2001). During multiplication on the insect, the conidia produce different virulence factors, identified as extracellular enzymes, especially proteases characterized as chymotrypsin-like serine proteinases with activity on casein, hide protein azure (HPA), azocoll, elastin Congo Red and collagen substrates (Chrzanowska et al., 2001), chitinases (Samsinakova et al., 1971; St-Leger et al., 1986) and lipases (Giraldo-Cardozo et al., 2001).

The production of extracellular enzymes was related to the infection process of the insects because it allowed the penetration of the germ tube across the cuticle which is formed by nearly 70% of proteins (Hepburn, 1985), chitin and lipids (Bidochka and Khachatourians, 1992), and the growth of the fungus through the use of hemolymph nutrients (Shimizu et al., 1993). The entomopathogenic fungus can diversify the production of virulence factors in response to the different insects (Moino et al., 1998). Inducer substrates have been tested in order to increase the protein content and to release the extracellular proteases to make easier the studies on kinetic activity and structure and to correlate with virulence levels of the fungal strain in bioassays on insects. Neves and Hirose (2005) observed that the Brazilian B. bassiana strain CG432 bioassayed on coffee berry borer, Hypothenemus hampei, presented better conidia production and more than 70% of mortality, with 10⁸ conidia/mL concentration among 61 Brazilian strains when reactivated on the same insect. Previous studies showed that extracelullar proteases produced by this strain were active at pH 4.5, 7.0 and 9.5, suggesting the production at least three different type of proteases (Stürmer et al., 2003/2004).

There are many papers about protease production and their characteristics, but more information is necessary about B. bassiana strains adapted at Brazilian conditions. Thus, the objective of this study was to determine the culture time for the maximum production of extracellular proteases by B. bassiana CG432 reactivated on coffee berry borer and the effect of some reaction and storage conditions on the activity and stability of these enzymes.

MATERIALS AND METHODS

Fungal strain

B. bassiana CG432 was isolated from adult insects of the family Membracidae (Homoptera), Rio Grande do Norte State, northeast, Brazil, supplied to the Entomopathogen Bank in the Department of Agronomy at Londrina State University by the Brazilian Corporation for Agricultural Research _ EMBRAPA/Cenargen.

Conidium reactivation

The fungus conidia was maintained in Petri dishes containing the solid culture medium for B. bassiana: 20g agar, 10g anhydrous D-glucose, 5g yeast extract, 1.58g NaNO₃, 1.05g Na₂HPO₄.7H₂O, 1g KCl; 0.6g MgSO₄.7H₂O and 0.36g KH₂PO₄, distilled water to one liter (Alves, 1998), at 25°C, 12h photophase, for ten days. Media produced conidia were sprayed on H. hampei alive adults previously treated with 0.5% sodium hypochlorite solution placed on Petri dishes and incubated at 25ºC until insect death. Newly emerged conidia from the insect were subcultured not more than four times, at ten days intervals in solid culture medium and used to prepare the reactivated inoculum suspension containing 10⁸conidia/mL (Neves and Hirose, 2005).

Fungal growth conditions

The fungus was cultivated at 28ºC, 150 rpm during for nine days in 250 mL erlenmeyers flacks, with 100 mL of liquid culture medium (as above culture medium) without agar with 1% of the suspension of 10⁸conidia/mL as the inoculum, in duplicate. One un-inoculated culture medium was used as control. At 24 h, a group of the three flacks (duplicate and control) was filtrated through previously weighed Whatman ! 1 filter papers and the filtrate, called crude extract (CE), was dialysed against 5 mM phosphate buffer pH 7 at 4ºC, served as the enzyme source for determination of the extracellular protease activity and the soluble protein content.

Biomass determination

Biomass was determined from the residue material on the filter paper. The biomass obtained from the 1^st to the 9^th day of cultivation was washed with two aliquots (50 mL each) of distilled water and dried at 70ºC until constant weight.

Extracellular protease assay

Extracellular protease was determinated (Ito, 2003), using 200µl aliquots of CEs obtained from the first to the ninth day of culture were incubated at 37ºC for 30 minutes with 100µl of the 50mM phosphate buffer pH 7 and 250µL bovine serum albumin 5mg/mL dissolved in the same buffer. The reaction was interrupted with 250mL 10% TCA and centrifuged at 1100g for 15 minutes The protease activity was considered as the absorbance lecture at 650 nm corresponding to the released soluble peptide content of the supernatant (Hartree, 1972). Each 0.1 increased in absorbance was considered as one unity of protease activity.

Effect of temperature and íons Cl^- on protease activity

Temperature and Cl^- effect on protease activity was determined using the CE obtained after 5 days of cultivation in 50 mM phosphate buffer pH 7 at temperatures from 20 to 70°C. The effect of 0.1 to 1.0 M NaCl concentration was determinated at 37ºC in 50 mM acetate, phosphate and Glycine-NaOH buffer at pH 4.5, 7 and 9.5, respectively (Stürmer et al., 2003/2004).

Protease stability on storage conditions

Protease stability was evaluated by determining the protease activity of CE obtained on the 5^th cultivation day after stay storing at 4ºC or frozen at -18ºC for 30 days. The effect of successive freezing and thawing of the sample was also evaluated 6 times (each 5 days) during the same time period.

RESULTS AND DISCUSSION

Protease production and fungal growth

The highest extracellular protease activity by B bassiana CG432 (Fig. 1A) was observed on the 5^th day (Abs 650 nm 0.489) but around 80% of this activity was obtained after 2^nd day of the cultivation. After the 5^th day, there was an abrupt reduction of the activity until the 9^th day. Variable time courses of extracellular protease production have been related in literature, reflecting the variability in protease production on different media. The stimulatory effects of a series of 24 nitrogen sources including inorganic, organic nonprotein, proteins and complex natural media on the production of proteases in submerged cultures demonstrated that the maximum amount of protease into the maize meal, yeast extract, ground maize and wheat bran broth was released 3 days after inoculation (Kucera, 1971).

Others studies that used inducer substrates to produce proteases from B. bassiana, stopped the cultures in between 3^rd to 6^th day of the cultivation as 0.2% lyophilized porcine blood plasma and 0.5% ground larval of Apis mellifera with higher release at 3^rd day by B. Bassiana 278 (Chrzanowska et al., 2001), 1% gelatin at 3^rd and 4^th day by B bassiana GK2016 (Bidochka and Khachatourians, 1987 and 1988), 1% gelatin and casein, at 4^th day by B. bassiana 11892A (Urtz and Rice, 2000), ground migratory grasshopper cuticle, at 5^th day by B bassiana GK2016 and GK2018 (Bidochka and Khachatourians, 1990, 1991, 1993), colloidal chitin, at 6^th day (Havukkala et al. 1993), and liquid medium with cuticle ground from the own coffee berry borer, Hypothenemus hampei at 5^th to 7^th days after the inoculation (Giraldo-Cardozo et al., 2001).

The biomass production (Fig. 1B) and the concentration of soluble protein (Fig. 1C) of the culture medium increased after 2^nd day and coincided with 80% of the total of the extracellular protease production, after a small lag period of 24 h shorter than obtained by two isolates of B. Bassiana (Arcas et al., 1999). These results could be related with the production of complex proteolityc enzymes during the reactivation of the inoculum on coffee berry borer alive, using liquid medium without inducer substrates such as the cuticle (Havukkala et al. 1993; Urtz and Rice, 2000) or extract from insects (Bidochka and Khachatourians, 1990, 1991, 1993; Giraldo-Cardozo et al., 2001).

The active growth continued until the 5^th day when the enzyme was released in the culture medium reaching the maximum extracellular protease activity. After that, the biomass production was stable when the protease release was reduced (Fig. 1A and 1B). These results confirmed the importance of the extracellular protease activity on the substrate utilization during the multiplication celular phase of this fungus as related to other microorganisms (Fu-Chu et al., 2004). The concentration of soluble protein of the culture medium was highest on the 7^th day probably related with biomass (Fig. 1C).

The previous reactivation of the fungus on insects proposed in this work presented the advantage of less laborious laboratory work to produce the inoculum than to prepare different substrates and less cultivation time, considering that 80% of the total activity was obtained on 2^nd day.

Temperature effect

The extracellular proteases were more active between 40 and 60ºC (Fig. 2). This result was better than those by Bidochka and Khachatourians (1987), with optimum temperature between 37 to 42ºC.

Cl^- ion concentration effect

The CE proteases remained active when analyzed in increasing concentrations of chloride ions of 0.1 to 1.0 M at pH 4.5, 7.0 and 9.5 (Fig. 3). This could be important information, because chloride ions are frequently used on buffer analysis and purification laboratory procedures.

Protease stability in storage

It was observed that the proteases maintained above 90% of their activity up to the 30^ty day under freezing and above 80% until the 15^th day under refrigeration. The activity fell down to 50% at the 5^th times of freezing/thawing (Fig. 4). Thus, repetitive freezing of the extracts should be avoided.

At least, the extracellular proteases produced in these conditions presented optimum temperature activity at 60°C, without interferences from Cl^- ion and was stable for many days on refrigeration or freezing conditions. This fact could be very important in purification procedures. Also, proteases produced under same conditions were stable during 10 days at 25ºC (Stürmer et al., 2003/2004) being too important on insect procedures for bioassay when used as bioinseticide.

ACKNOWLEDGMENTS

Ito, E. T. whishes to thank Fundação Araucária/Pr and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the fellowship for the Master Course in Biotechnology. The authors acknowledge Pro-Reitoria de Pesquisa e Pós-Graduação (PROPPG-UEL-number 24698/2003), Fundação Araucária (number-213/2003) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-number 473595/2003-6) for the financial supports and to Profª Drª Maria Helena Pimenta Pinotti for the English revision.

Received: July 01, 2005;

Revised: January 09, 2006;

Accepted: October 23, 2006

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Author for correspondence

Publication Dates

• Publication in this collection
  12 June 2007
• Date of issue
  Mar 2007

History

• Accepted
  23 Oct 2006
• Reviewed
  09 Jan 2006
• Received
  01 July 2005